Articles of manufacture

ABSTRACT

The present invention relates to highly selective phosphodiesterase (PDE) enzyme inhibitors and to their use in pharmaceutical articles of manufacture. In particular, the present invention relates to potent inhibitors of cyclic guanosine 3′,5′-monophosphate specific phosphodiesterase type 5 (PDE5) that when incorporated into a pharmaceutical product at about 1 to about 20 mg unit dosage are useful for the treatment of sexual dysfunction. The articles of manufacture described herein are characterized by selective PDE5 inhibition, and accordingly, provide a benefit in therapeutic areas where inhibition of PDE5 is desired, with minimization or elimination of adverse side effects resulting from inhibition of other phosphodiesterase enzymes.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of provisional patentapplication Serial No. 60/132,036, filed Apr. 30, 1999.

FIELD OF THE INVENTION

[0002] The present invention relates to highly selectivephosphodiesterase (PDE) enzyme inhibitors and to their use inpharmaceutical articles of manufacture. In particular, the presentinvention relates to potent inhibitors of cyclic guanosine3′,5′-monophosphate specific phosphodiesterase type 5 (PDE5) that whenincorporated into a pharmaceutical product are useful for the treatmentof sexual dysfunction. The articles of manufacture described herein arecharacterized by selective PDE5 inhibition, and accordingly, provide abenefit in therapeutic areas where inhibition of PDE5 is desired, withminimization or elimination of adverse side effects resulting frominhibition of other phosphodiesterase enzymes.

BACKGROUND OF THE INVENTION

[0003] The biochemical, physiological, and clinical effects of cyclicguanosine 3′,5′-monophosphate specific phosphodiesterase (cGMP-specificPDE) inhibitors suggest their utility in a variety of disease states inwhich modulation of smooth muscle, renal, hemostatic, inflammatory,and/or endocrine function is desired. Type 5 cGMP-specificphosphodiesterase (PDE5) is the major CGMP hydrolyzing enzyme invascular smooth muscle, and its expression in penile corpus cavernosumhas been reported (Taher et al., J. Urol., 149:285A (1993)). Thus, PDE5is an attractive target in the treatment of sexual dysfunction (Murray,DN&P 6(3):150-56 (1993)).

[0004] A pharmaceutical product, which provides a PDE5 inhibitor, iscurrently available and marketed under the trademark VIAGRA®. The activeingredient in VIAGRA® is sildenafil. The product is sold as an articleof manufacture including 25, 50, and 100 mg tablets of sildenafil and apackage insert. The package insert provides that sildenafil is a morepotent inhibitor of PDE5 than other known phosphodiesterases (greaterthan 80 fold for PDE1 inhibition, greater than 1,000 fold for PDE2,PDE3, and PDE4 inhibition). The IC₅₀ for sildenafil against PDE5 hasbeen reported as 3 nM (Drugs of the Future, 22(2), pp. 128-143 (1997)),and as 3.9 nM (Boolell et al., Int. J. of Impotence Res., 8 p. 47-52(1996)) Sildenafil is described as having a 4,000-fold selectivity forPDE5 versus PDE3, and only a 10-fold selectivity for PDE5 versus PDE6.Its relative lack of selectivity for PDE6 is theorized to be the basisfor abnormalities related to color vision.

[0005] While sildenafil has obtained significant commercial success, ithas fallen short due to its significant adverse side effects, includingfacial flushing (10% incidence rate). Adverse side effects limit the useof sildenafil in patients suffering from visual abnormalities,hypertension, and, most significantly, by individuals who use organicnitrates (Welds et al., Amer. J. of Cardiology, 83 (5A), pp. 21(C)-28(C) (1999)).

[0006] The use of sildenafil in patients taking organic nitrates isbelieved to cause a clinically significant drop in blood pressure whichcould place the patient in danger. Accordingly, the package label forsildenafil provides strict contraindications against its use incombination with organic nitrates (e.g., nitroglycerin, isosorbidemononitrate, isosorbide dinitrate, erythrityl tetranitrate) and othernitric oxide donors in any form, either regularly or intermittently,because sildenafil potentiates the hypotensive effects of nitrates. SeeC. R. Conti et al., Amer. J. of Cardiology, 83(5A), pp. 29C-34C (1999).Thus, even with the availability of sildenafil, there remains a need toidentify improved pharmaceutical products that are useful in treatingsexual dysfunction.

[0007] The present invention provides an article of manufacture forhuman pharmaceutical use, comprising a package insert, a container, andan oral dosage form comprising a selective PDE5 inhibitor at unitdosages between about 1 and about 20 mg/dosage form. The beneficialeffects of the present invention were observed in clinical studies andthrough the discovery that a selective PDE5 inhibitor meeting thefollowing criteria allows for the effective oral administration of about1 to about 20 mg/dosage form without contraindications generallyrequired for PDE5 inhibitor products, such as warnings directed tovision abnormalities. A selective PDE5 inhibitor of the presentinvention exhibits:

[0008] 1) at least a 100 fold differential in the IC₅₀ values for theinhibition of PDE5 versus PDE6 for a particular PDE5 inhibitor (i.e.,the IC₅₀ value versus PDE5 is at least 100 times less than the IC₅₀value versus PDE6);

[0009] 2) at least a 1000 fold differential in the IC₅₀ values for theinhibition of PDE5 versus PDE1c; and

[0010] 3) an IC₅₀ value less than 10 nM.

[0011] Significantly, clinical studies also revealed that an effectiveproduct having a reduced tendency to cause flushing in susceptibleindividuals can be provided. Most unexpectedly, the product also can beadministered with clinically insignificant side effects associated withthe combined effects of a PDE5 inhibitor and an organic nitrate. Thus,the contraindication once believed necessary for a product containing aPDE5 inhibitor is unnecessary when a selective PDE5 inhibitor, asdefined above, is used as disclosed herein. Thus, the present inventionprovides an effective therapy for sexual dysfunction in individuals whopreviously were untreatable or suffered from unacceptable side effects,including individuals having cardiovascular disease, such as inindividuals requiring nitrate therapy, having suffered a myocardialinfarction more than three months before the onset of sexual dysfunctiontherapy, and suffering from class 1 congestive heart failure as definedby the New York Heart Association (NYHA), or individuals suffering fromvision abnormalities.

SUMMARY OF THE INVENTION

[0012] The present invention provides an article of manufacture forhuman pharmaceutical use, comprising a package insert, a container, andan oral dosage form comprising about 1 to about 20 mg of a selectivePDE5 inhibitor per dosage form.

[0013] The present invention further provides a method of treatingconditions where inhibition of PDE5 is desired, which comprisesadministering to a patient in need thereof an oral dosage formcontaining about 1 to about 20 mg of a selective PDE5 inhibitor, asneeded, up to a total dose of 20 mg/day. The invention further providesthe use of an oral dosage form comprising a selective PDE5 inhibitor ata dosage of about 1 to about 20 mg for the treatment of sexualdysfunction.

[0014] Specific conditions that can be treated by the method and articleof the present invention, include, but are not limited to, male erectiledysfunction and female sexual dysfunction, particularly female arousaldisorder, also known as female sexual arousal disorder.

[0015] In particular, the present invention provides an article ofmanufacture for human pharmaceutical use comprising:

[0016] (a) an oral dosage form comprising about 1 to about 20 mg of aselective PDE5 inhibitor having

[0017] (i) at least a 100 fold differential in IC₅₀ values for theinhibition of PDE5 versus PDE6,

[0018] (ii) at least a 1000 fold differential in IC₅₀ values for theinhibition of PDE5 versus PDE1c,

[0019] (iii) an IC₅₀ less than 10 nM, and

[0020] (iv) sufficient bioavailability to be effective in about 1 toabout 20 mg unit oral dosages;

[0021] (b) a package insert providing that the PDE5 inhibitor is usefulto treat sexual dysfunction in a patient in need thereof, and that isfree of contradictions associated with administration of organicnitrates; and

[0022] (c) a container.

[0023] The present invention further provides an article of manufacturefor human pharmaceutical use comprising:

[0024] (a) an oral dosage form comprising about 1 to about 20 mg ofselective PDE5 inhibitor having

[0025] (i) at least a 100 fold differential in IC₅₀ values for theinhibition of PDE5 versus PDE6,

[0026] (ii) at least a 1000 fold differential in IC₅₀ values for theinhibition of PDE5 versus PDE1c,

[0027] (iii) an IC₅₀ less than 10 nM, and

[0028] (iv) a sufficient bioavailability to be effective in about 1 toabout 20 mg unit oral dosages;

[0029] (b) a package insert providing that the PDE5 inhibitor is usefulto treat sexual dysfunction in a patient in need thereof and that isusing an organic nitrate; and

[0030] (c) a container.

[0031] The present invention also provides an article of manufacture forhuman pharmaceutical use comprising:

[0032] (a) an oral dosage form comprising about 1 to about 20 mg of aselective PDE5 inhibitor having

[0033] (i) at least a 100 fold differential in IC₅₀ values for theinhibition of PDE5 versus PDE6,

[0034] (ii) at least 1000 fold differential in IC₅₀ values for theinhibition of PDE5 versus PDE1c,

[0035] (iii) an IC₅₀ less than 10 nM, and

[0036] (iv) a sufficient bioavailability to be effective in about 1 toabout 20 mg unit oral dosages;

[0037] (b) a package insert providing that the PDE5 inhibitor is usefulto treat sexual dysfunction in a patient in need thereof and that issuffering from a condition selected from the group consisting of aretinal disease, proneness to flushing, proneness to visionabnormalities, class 1 congestive heart failure, a myocardial infarction90 days or more before onset of the sexual dysfunction treatment, andcombinations thereof; and

[0038] (c) a container.

DETAILED DESCRIPTION

[0039] For purposes of the present invention as disclosed and describedherein, the following terms and abbreviations are defined as follows.

[0040] The term “container” means any receptacle and closure thereforsuitable for storing, shipping, dispensing, and/or handling apharmaceutical product.

[0041] The term “IC₅₀” is the measure of potency of a compound toinhibit a particular PDE enzyme (e.g., PDE1c, PDE5, or PDE6). The IC₅₀is the concentration of a compound that results in 50% enzyme inhibitionin a single dose-response experiment. Determining the IC₅₀ value for acompound is readily carried out by a known in vitro methodologygenerally described in Y. Cheng et al., Biochem. Pharmacol., 22, pp.3099-3108 (1973).

[0042] The term “package insert” means information accompanying theproduct that provides a description of how to administer the product,along with the safety and efficacy data required to allow the physician,pharmacist, and patient to make an informed decision regarding use ofthe product. The package insert generally is regarded as the “label” fora pharmaceutical product.

[0043] The term “oral dosage form” is used in a general sense toreference pharmaceutical products administered orally. Oral dosage formsare recognized by those skilled in the art to include such forms asliquid formulations, tablets, capsules, and gelcaps.

[0044] The term “selective PDE5 inhibitor” is defined as a PDE5inhibitor having:

[0045] 1) an IC₅₀ value for the inhibition of PDE5 at least 100 timesless than the IC₅₀ value for the inhibition of PDE6;

[0046] 2) an IC₅₀ value for the inhibition of PDE5 at least 1,000 timesless than the IC₅₀ value for the inhibition of PDE1c; and

[0047] 3) an IC₅₀ value for the inhibition of PDE5 less than 10 nM.

[0048] Selective PDE5 inhibitors vary significantly in chemicalstructure, and their use in the present invention is not dependent onchemical structure, but rather on the selectivity and potency parametersdisclosed herein.

[0049] The term “vision abnormalities” means abnormal visioncharacterized by blue-green vision believed to be caused by PDE6inhibition.

[0050] The term “flushing” means an episodic redness of the face andneck attributed to vasodilation caused by the ingestion of a drug,usually accompanied by a feeling of warmth over the face and neck andsometimes accompanied by perspiration.

[0051] The term “free drug” means solid particles of drug not intimatelyembedded in a polymeric coprecipitate.

[0052] As previously stated, the present invention is directed to anarticle of manufacture for human pharmaceutical use, comprising apackage insert, a container, and a dosage form comprising about 1 toabout 20 mg of a selective PDE5 inhibitor per unit dosage form. Aselective PDE5 inhibitor useful in the present invention is a PDE5inhibitor having:

[0053] 1) at least a 100 fold differential in IC₅. values for theinhibition of PDE5 versus PDE6;

[0054] 2) at least a 1000 fold differential in IC₅₀ values for theinhibition of PDE5 versus PDE1c; and

[0055] 3) an IC₅₀ value less than 10 nM; and is sufficientlybioavailable to be effective in about 1 to about 20 mg unit dosages.

[0056] The differential is expressed as a PDE6/PDE5 ratio of IC₅₀values, i.e., the ratio of the IC₅₀ value versus PDE6 to the IC₅₀ valueversus PDE5 (PDE6/PDE5) is greater than 100, more preferably greaterthan 300, and most preferably greater than 500.

[0057] Similarly, the ratio of IC₅₀ value versus PDE1c to IC₅₀ valueversus PDE5 (PDE1c/PDE5) is greater than 1000. Preferred PDE5 inhibitorshave a greater than 3,000 fold differential between the inhibition ofPDE5 and PDE1c, more preferably greater than a 5,000 fold differentialbetween IC₅₀ value versus PDE5 and PDE1c. The potency of the inhibitor,as represented by the IC₅₀ value versus PDE5, is less than 10 nM,preferably less than 5 nM, more preferably less than 2 nM, and mostpreferably less than 1 nM.

[0058] The package insert provides a description of how to administer apharmaceutical product, along with the safety and efficacy data requiredto allow the physician, pharmacist, and patient to make an informeddecision regarding the use of the product. The package insert generallyis regarded as the label of the pharmaceutical product. The packageinsert incorporated into the present article of manufacture indicatesthat the selective PDE5 inhibitor is useful in the treatment ofconditions wherein inhibition of PDE5 is desired. The package insertalso provides instructions to administer one or more about 1 to about 20mg unit dosage forms as needed, up to a maximum total dose of 20 mg perday. Preferably, the dose administered is about 5 to about 20 mg/day,more preferably about 5 to about 15 mg, and most preferably an about 5mg or about 10 mg dosage form administered once per day, as needed.

[0059] Preferred conditions to be treated include sexual dysfunction(including male erectile dysfunction; and female sexual dysfunction, andmore preferably female arousal disorder (FAD)). The preferred conditionto be treated is male erectile dysfunction.

[0060] Significantly, the package insert supports use of the product totreat sexual dysfunction in patients suffering from a retinal disease,for example diabetic retinopathy or retinitis pigmentosa, or in patientswho are using organic nitrates. Thus, the package insert preferably isfree of contraindications associated with these conditions, andparticularly the administration of the dosage form with an organicnitrate. More preferably, the package insert also is free of anycautions or warnings both associated with retinal diseases, particularlyretinitis pigmentosa, and associated with individuals prone to visionabnormalities. Preferably, the package insert also reports incidences offlushing below 2%, preferably below 1%, and most preferably below 0.5%,of the patients administered the dosage form. The incidence rate offlushing demonstrates marked improvement over prior pharmaceuticalproducts containing a PDE5 inhibitor.

[0061] The container used in the present article of manufacture isconventional in the pharmaceutical arts. Generally, the container is ablister pack, foil packet, glass or plastic bottle and accompanying capor closure, or other such article suitable for use by the patient orpharmacist. Preferably, the container is sized to accommodate 1-1000solid dosage forms, preferably 1 to 500 solid dosage forms, and mostpreferably, 5 to 30 solid dosage forms.

[0062] Oral dosage forms are recognized by those skilled in the art toinclude, for example, such forms as liquid formulations, tablets,capsules, and gelcaps. Preferably the dosage forms are solid dosageforms, particularly, tablets comprising about 1 to about 20 mg of aselective PDE5 inhibitor. Any pharmaceutically acceptable excipients fororal use are suitable for preparation of such dosage forms. Suitablepharmaceutical dosage forms include coprecipitate forms described, forexample, in Butler U.S. Pat. No. 5,985,326, incorporated herein byreference. In preferred embodiments, the unit dosage form of the presentinvention is a solid free of a coprecipitate form of the PDE5 inhibitor,but rather contains a solid PDE5 inhibitor as a free drug.

[0063] Preferably, the tablets comprise pharmaceutical excipientsgenerally recognized as safe such as lactose, microcrystallinecellulose, starch, calcium carbonate, magnesium stearate, stearic acid,talc, and colloidal silicon dioxide, and are prepared by standardpharmaceutical manufacturing techniques as described in Remington'sPharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa.(1990). Such techniques include, for example, wet granulation followedby drying, milling, and compression into tablets with or without filmcoating; dry granulation followed by milling, compression into tabletswith or without film coating; dry blending followed by compression intotablets, with or without film coating; molded tablets; wet granulation,dried and filled into gelatin capsules; dry blend filled into gelatincapsules; or suspension and solution filled into gelatin capsules.Generally, the solid dosage forms have identifying marks which aredebossed or imprinted on the surface.

[0064] The present invention is based on detailed experiments andclinical trials, and the unexpected observations that side effectspreviously believed to be indicative of PDE5 inhibition can be reducedto clinically insignificant levels by the selection of a selective PDE5inhibitor having the specific characteristics outlined herein, namely:

[0065] 1) at least a 100 fold differential in the IC₅₀ values for theinhibition of PDE5 versus PDE6;

[0066] 2) at least a 1000 fold differential in the IC₅₀ values for theinhibition of PDE5 versus PDE1c; and

[0067] 3) an IC₅₀ value for the inhibition of PDE5 less than 10 nM.

[0068] This unexpected observation enabled the development of articlesof manufacture that incorporate a selective PDE5 inhibitor in about 1 toabout 20 mg unit dosage forms that, when orally administered, minimizeundesired side effects previously believed unavoidable. These sideeffects include facial flushing, vision abnormalities, and a significantdecrease in blood pressure when the PDE5 inhibitor is administered aloneor in combination with an organic nitrate. The minimal effect of apresent PDE5 inhibitor, administered in about 1 to about 20 mg unitdosage forms, on PDE6 also allows the administration of a selective PDE5inhibitor to patients suffering from a retinal disease, like diabeticretinopathy or retinitis pigmentosa.

[0069] One such selective PDE5 inhibitor, i.e.,(6R-trans)-6-(1,3-benzodioxol-5-yl)-2,3,6,7,12,12a-hexahydro-2-methylpyrazino[1′,2′:1,6]pyrido[3,4-b]indole-1,4-dione,alternatively named(6R,12aR)-2,3,6,7,12,12a-hexahydro-2-methyl-6-(3,4-methylenedioxyphenyl)pyrazino[2′,1′:6,1]pyrido[3,4-b]indole-1,4-dione,is disclosed in Daugan U.S. Pat. No. 5,859,006, and represented bystructural formula (I):

[0070] The compound of formula (I) was demonstrated in human clinicalstudies to exert a minimal impact on systolic blood pressure whenadministered in conjunction with organic nitrates. By contrast,sildenafil, when administered with nitrates, demonstrates as much as afour-fold greater decrease in systolic blood pressure over a placebo,which leads to the contraindications in the VIAGRA® insert, and inwarnings to certain patients.

[0071] Selective PDE5 inhibitors vary significantly in chemicalstructure, and the use of a selective PDE5 inhibitor as defined in thepresent invention is not dependent on a particular chemical structure,but rather on the critical parameters outlined herein. However,preferred compounds having the required potency and selectivity can bereadily identified by tests described herein from those described inDaugan U.S. Pat. No. 5,859,006, Daugan et al. U.S. Pat. No. 5,981,527,and Daugan et al. U.S. Pat. No. 6,001,847, each of which is incorporatedherein by reference.

[0072] Preferred compounds of Daugan U.S. Pat. No. 5,859,006 and Dauganet al. U.S. Pat. No. 5,981,527 are represented by structural formula(II):

[0073] wherein

[0074] R⁰ is selected from the group consisting of hydrogen, halogen,and C₁₋₆alkyl;

[0075] R¹ is selected from the group consisting of hydrogen, C₁₋₆alkyl,C₂₋₆alkenyl, C₂₋₆alkynyl, halo-C₁₋₆alkyl, C₃₋₈cycloalkyl,C₃₋₈cycloalkylC₁₋₃alkyl, arylC₁₋₃alkyl, wherein aryl is phenyl or phenylsubstituted with one to three substituents selected from the groupconsisting of halogen, C₁₋₆alkyl, C₁₋₆alkoxy, methylenedioxy, andmixtures thereof, and heteroarylC₁₋₃alkyl, wherein heteroaryl isthienyl, furyl, or pyridyl, each optionally substituted with one tothree substituents selected from the group consisting of halogen,C₁₋₆alkyl, C₁₋₆alkoxy, and mixtures thereof;

[0076] R² represents an optionally substituted monocyclic aromatic ringselected from benzene, thiophene, furan, and pyridine, or an optionallysubstituted bicyclic ring

[0077]  attached to the rest of the molecule via one of the benzene ringcarbon atoms and wherein the fused ring A is a 5- or 6-membered ring,saturated or partially or fully unsaturated, and comprises carbon atomsand optionally one or two heteroatoms selected from the group consistingof oxygen, sulphur and nitrogen;

[0078] R³ represents hydrogen or C₁₋₃alkyl, or R¹ and R³ togetherrepresent a 3- or 4-membered alkyl or alkenyl chain; and salts andsolvates thereof.

[0079] Other preferred compounds are those of formula (II) wherein:

[0080] R⁰ is hydrogen, halogen, or C₁₋₆alkyl;

[0081] R¹ is hydrogen or C₁₋₆alkyl;

[0082] R² is the bicyclic ring

[0083]  which can be optionally substituted by one or more groupsselected from halogen and C₁₋₃alkyl; and

[0084] R³ is hydrogen or C₁₋₃alkyl.

[0085] The following Table 1 illustrates PDE5 and PDE6 IC₅₀ values forrepresentative selective PDE5 inhibitors disclosed in U.S. Pat. No.5,859,006, as determined by the procedures described herein. TABLE 1Compound PDE5 IC₅₀ (nM) PDE6 IC₅₀ (nM) PDE6/PDE5 1 5 663 133 2 2 937 4693 2 420 210 4 5 729 146 5 2.5 3400 1360

[0086] Compound 5 in Table 1 has the structural formula (I) andadditionally demonstrates an IC₅o against PDE1c of 10,000 and a ratio ofPDE1c/PDE5 of 4,000.

[0087] The structures of Compound Nos. 1-5 in Table 1 are as instructural formula (II) wherein R⁰, R¹, R², and R³ are as follows:Compound R⁰ R¹ R² R³ 1 H

H 2 H CH₃

H 3 H

H 4 H H

CH₃ 5 H CH₃

H

[0088] The data in Table 1 indicate that a compound of structuralformula (I), wherein R¹ is hydrogen or C₁₋₆alkyl, R² is

[0089] and R³ is hydrogen is especially preferred. Preferably, A is

[0090] Preferred compounds are:(6R,12aR)-2,3,6,7,12,12a-hexahydro-2-methyl-6-(3,4-methylenedioxyphenyl)pyrazino[2′,1′:6,1]pyrido[3,4-b]indole-1,4-dione;and(3s,6R,12aR)-2,3,6,7,12,12a-hexahydro-2,3-dimethyl-6-(3,4-methylenedioxyphenyl)pyrazino[2′,1′:6,1]-pyrido[3,4-b]indole-1,4-dione;and physiologically acceptable salts and solvates (e.g., hydrates)thereof.

[0091] Other exemplary compounds useful in the present invention arethose disclosed in Daugan et al. U.S. Pat. No. 6,001,847 and WO97/43287, incorporated herein by reference.

[0092] Further exemplary compounds for use in the present invention aredisclosed PCT application PCT/EP98/06050, which designates the U.S.,entitled “Chemical Compounds,” inventors A. Bombrun and F. Gellibert,the disclosure of which is specifically incorporated herein byreference. This class of compounds has the structural formula (III):

[0093] and salts and solvates (e.g., hydrates) wherein

[0094] C represents a 5- or 6-membered heteroaryl group containing atleast one heteroatom selected from the group consisting of oxygen,nitrogen, and sulfur;

[0095] R¹² represents hydrogen or halogen;

[0096] R¹³ is selected from the group consisting of

[0097] hydrogen,

[0098] nitro (NO₂),

[0099] trifluoromethyl,

[0100] trifluoromethoxy,

[0101] halogen,

[0102] cyano (CN),

[0103] a 5- or 6-membered heterocyclic group containing at least oneheteroatom selected from the group consisting of oxygen, nitrogen, andsulphur, optionally substituted with C(═O)OR^(a) or C₁₄alkyl,

[0104] C₁₆alkyl optionally substituted with OR^(h),

[0105] C₁₋₃alkoxy,

[0106] C(═O)R^(h),

[0107] OC(═O)OR^(h),

[0108] C(=O)OR^(h),

[0109] C₁₋₄alkyleneHet,

[0110] C₁₄alkyleneC(═O)OR^(h),

[0111] OC₁₋₄alkyleneC(═O)OR^(h),

[0112] C₁₋₄alkyleneOC₁₋₄alkyleneC(═O) OR^(h),

[0113] C(═O)NR^(i)SO₂R^(j),

[0114] C(═O)C₁₋₄alkyleneHet,

[0115] C₁₋₄alkyleneNR^(h)R^(i),

[0116] C₂₋₆alkenyleneNR^(h)R^(i),

[0117] C(═O)NR^(h)R^(i),

[0118] C(═O)NR^(h)R^(i),

[0119] C(═O)NR^(h)C₁₋₄alkyleneOR^(i),

[0120] C(═O)NR^(h)C₁₋₄alkyleneHet,

[0121] OR^(i),

[0122] OC₂₋₄alkyleneNR^(h)R^(i),

[0123] OC₁₋₄alkyleneCH(OR^(h))CH₂NR^(h)R^(i),

[0124] OC₁₋₄alkyleneHet,

[0125] OC₂₋₄alkyleneOR^(h),

[0126] OC₂₋₄alkyleneNR^(h)C(═O) OR^(h),

[0127] NR^(h)R^(i),

[0128] NR^(h)C₁₋₄alkyleneNR^(h)R^(i),

[0129] NR^(h)C(═O)R^(i),

[0130] NR^(h)C(═O)NR^(h)R^(i),

[0131] N(SO₂C₁₋₄alkyl)₂,

[0132] NR^(h)(SO₂C₁₋₄alkyl),

[0133] SO₂NR^(h)R^(i),

[0134] and OSO₂trifluoromethyl;

[0135] R¹⁴ is selected from the group consisting of hydrogen, halogen,OR^(h), C₁₋₆alkyl, NO₂, and NR^(h)R^(i);

[0136] or R¹³ and R¹⁴ are taken together to form a 3- or 4-memberedalkylene or alkenylene chain component of a 5- or 6-membered ring,optionally containing at least one heteroatom;

[0137] R¹⁵ is selected from the group consisting of hydrogen, halogen,NO₂, trifluoromethoxy, C₁₋₆alkyl, OC₁₋₆alkyl, and C(═O)OR^(h);

[0138] R¹⁶ is hydrogen,

[0139] or R¹⁵ and R¹⁶ are taken together to form a 3- or 4-memberedalkylene or alkenylene chain component of a 5- or 6-membered ring,optionally containing at least one heteroatom;

[0140] Het represents a 5- or 6-membered heterocyclic group containingat least one heteroatom selected from the group consisting of oxygen,nitrogen, and sulfur, and optionally substituted with C₁₋₄alkyl;

[0141] R^(h) and R^(i) can be the same or different and areindependently selected from hydrogen and C₁₋₆alkyl;

[0142] R^(j) represents phenyl or C₄₋₆cycloalkyl, wherein the phenyl orC₄₋₆cycloalkyl can be optionally substituted with one or more halogenatoms, one or more C(═O)OR^(h), or one or more OR^(h);

[0143] n is an integer 1, 2, or 3; and

[0144] m is an integer 1 or 2.

[0145] Preparations

[0146] Human PDE5 Preparation

[0147] Recombinant production of human PDE5 was carried out essentiallyas described in Example 7 of U.S. Pat. No. 5,702,936, incorporatedherein by reference, except that the yeast transformation vectoremployed, which is derived from the basic ADH2 plasmid described in V.Price et al., Methods in Enzymology, 1985, pages 308-318 (1990),incorporated yeast ADH2 promoter and terminator sequences rather thanADH1 promoter and terminator sequences and the Saccharomyces cerevisiasehost was the protease-deficient strain BJ2-54 deposited on Aug. 31, 1998with the American Type Culture Collection, Manassas, Va., underaccession number ATCC 74465. Transformed host cells were grown in 2×SC-leu medium, pH 6.2, with trace metals, and vitamins. After 24 hours,YEP medium containing glycerol was added to a final concentration of 2×YEP/3% glycerol. Approximately 24 hours later, cells were harvested,washed, and stored at −70° C.

[0148] Cell pellets (29 g) were thawed on ice with an equal volume oflysis buffer (25 mM Tris-Cl, pH 8, 5 mM MgCl₂, 0.25 mM dithiothreitol, 1mM benzamidine, and 10 μM ZnSO₄). Cells were lysed in a microfluidizerwith N₂ at 20,000 psi. The lysate was centrifuged and filtered through0.45 μm disposable filters. The filtrate was applied to a 150 mL columnof Q Sepharose Fast Flow (Pharmacia). The column was washed with 1.5volumes of Buffer A (20 mM Bis-Tris Propane, pH 6.8, 1 mM MgCl₂, 0.25 mMdithiothreitol, 10 μM ZnSO₄) and eluted with a step gradient of 125 mMNaCl in Buffer A followed by a linear gradient of 125-1000 mM NaCl inBuffer A.

[0149] Active fractions from the linear gradient were applied to a 180mL hydroxyapatite column in Buffer B (20 mM Bis-Tris Propane (pH 6.8), 1mM MgCl₂, 0.25 mM dithiothreitol, 10 μM ZnSO₄, and 250 mM KCl). Afterloading, the column was washed with 2 volumes of Buffer B and elutedwith a linear gradient of 0-125 mM potassium phosphate in Buffer B.Active fractions were pooled, precipitated with 60% ammonium sulfate,and resuspended in Buffer C (20 mM Bis-Tris Propane, pH 6.8, 125 mMNaCl, 0.5 mM dithiothreitol, and 10 μM ZnSO₄). The pool was applied to a140 mL column of Sephacryl S-300HR and eluted with Buffer C. Activefractions were diluted to 50% glycerol and stored at −20° C. Theresultant preparations were about 85% pure by SDS-PAGE.

[0150] Assay for PDE Activity

[0151] Activity of PDE5 can be measured by standard assays in the art.For example, specific activity of any PDE can be determined as follows.PDE assays utilizing a charcoal separation technique were performedessentially as described in Loughney et al., (1996), The Journal ofBiological Chemistry, 271:796-806. In this assay, PDE5 activity converts[³²P]cGMP to [³²P]5′GMP in proportion to the amount of PDE5 activitypresent. The [³²P] 5′GMP then is quantitatively converted to free [³²P]phosphate and unlabeled adenosine by the action of snake venom5′-nucleotidase. Hence, the amount of [³²P] phosphate liberated isproportional to enzyme activity. The assay is performed at 30 C in a 100μL reaction mixture containing (final concentrations) 40 mM Tris-Cl (pH8.0), 1 μM ZnSO₄, 5 mM MgCl₂, and 0.1 mg/mL bovine serium albumin. PDE5is present in quantities that yield <30% total hydrolysis of substrate(linear assay conditions). The assay is initiated by addition ofsubstrate (1 mM [³²P]cGMP), and the mixture is incubated for 12 minutes.Seventy-five (75) μg of Crotalus atrox venom then is added, and theincubation is continued for 3 more minutes (15 minutes total). Thereaction is stopped by addition of 200 mL of activated charcoal (25mg/mL suspension in 0.1 M NaH₂PO₄, pH 4). After centrifugation (750× gfor 3 minutes) to sediment the charcoal, a sample of the supernatant istaken for radioactivity determination in a scintillation counter and thePDE5 activity is calculated. The preparations had specific activities ofabout 3 μmoles cGMP hydrolyzed per minute per milligram protein.

[0152] Bovine PDE6 Preparation

[0153] Bovine PDE6 was supplied by Dr. N. Virmaux, INSERM U338,Strasbourg. Bovine retinas were prepared as described by Virmaux et al.,FEBS Letters, 12(6), pp. 325-328 (1971) and see also, A. Sitaramayya etal., Exp. Eye Res., 25, pp. 163-169 (1977). Briefly, unless statedotherwise, all operations were done in the cold and in dim red light.Eyes were kept in the cold and in the dark for up to four hours afterslaughtering.

[0154] Preparation of bovine retinal outer segment (ROS) basicallyfollowed procedures described by Schichi et al., J. Biol. Chem., 224:529(1969). In a typical experiment, 35 bovine retinas were ground in amortar with 35 mL 0.066 M phosphate buffer, pH 7.0, made up to 40% withsucrose, followed by homogenization in a Potter homogenizer (20 up anddown strokes). The suspension was centrifuged at 25,000× g for 20minutes. The pellet was homogenized in 7.5 mL 0.006 M phosphate buffer(40% in sucrose), and carefully layered under 7.5 mL of phosphate buffer(containing no sucrose). Centrifugation was conducted in a swing-outrotor at 45,000× g for 20 minutes, and produced a pellet which is blackat the bottom, and also a red band at the interface 0.066 M.phosphate—40% sucrose/0.066 M phosphate (crude ROS). The red material atthe interface was removed, diluted with phosphate buffer, spun down to apellet, and redistributed in buffered 40% sucrose as described above.This procedure was repeated 2 or 3 times until no pellet was formed. Thepurified ROS was washed in phosphate buffer and finally spun down to apellet at 25,000× g for 20 minutes. All materials were then kept frozenuntil used.

[0155] Hypotonic extracts were prepared by suspending isolated ROS in 10mM Tris-Cl pH 7.5, 1 mM EDTA, and 1 mM dithioerythritol, followed bycentrifugation at 100,000× g for 30 minutes.

[0156] The preparation was reported to have a specific activity of about35 nmoles cGMP hydrolyzed per minute per milligram protein.

[0157] PDE1c Preparation from Spodoptera fugiperda Cells (Sf9)

[0158] Cell pellets (59) were thawed on ice with 20 ml of Lysis Buffer(50 mM MOPS pH 7.4, 10 μM ZnSO₄, 0.1 mM CaCl₂, 1 mM DTT, 2 mMbenzamidine HCl, 5 μg/ml each of pepstatin, leupeptin, and aprotenin).Cells were lysed by passage through a French pressure cell (SLM-Aminco)while temperatures were maintained below 10° C. The resultant cellhomogenate was centrifuged at 36,000 rpm at 4° C. for 45 minutes in aBeckman ultracentrifuge using a Type TI45 rotor. The supernatant wasdiscarded and the resultant pellet was resuspended with 40 ml ofSolubilization Buffer (Lysis Buffer containing 1M NaCl, 0.1M MgCl₂, 1 mMCaCl₂, 20 ug/ml calmodulin, and 1% Sulfobetaine SB12 (Z3-12) bysonicating using a VibraCell tuner with a microtip for 3×30 seconds.This was performed in a crushed ice/salt mix for cooling. Followingsonication, the mixture was slowly mixed for 30 minutes at 4° C. tofinish solubilizing membrane bound proteins. This mixture wascentrifuged in a Beckman ultracentrifuge using a type TI45 rotor at36,000 rpm for 45 minutes. The supernatant was diluted with Lysis Buffercontaining 10 μg/ml calpain inhibitor I and II. The precipitated proteinwas centrifuged for 20 minutes at 9,000 rpm in a Beckman JA-10 rotor.The recovered supernatant then was subjected to Mimetic Blue AP AgaroseChromatography.

[0159] In order to run the Mimetic Blue AP Agarose Column, the resininitially was shielded by the application of 10 bed volumes of 1%polyvinyl-pyrrolidine (i.e., MW of 40,000) to block nonspecific bindingsites. The loosely bound PVP-40 was removed by washing with 10 bedvolumes of 2M NaCl, and 10 mM sodium citrate pH 3.4. Just prior toaddition of the solubilized PDE1c sample, the column was equilibratedwith 5 bed volumes of Column Buffer A (50 mM MOPS pH 7.4, 10 μM ZnSO₄, 5mM MgCl₂, 0.1 mM CaCl₂, 1 mM DTT, 2 mM benzamidine HCl).

[0160] The solubilized sample was applied to the column at a flow rateof 2 ml/min with recycling such that the total sample was applied 4 to 5times in 12 hours. After loading was completed, the column was washedwith 10 column volumes of Column Buffer A, followed by 5 column volumesof Column Buffer B (Column Buffer A containing 20 mM 5′-AMP), andfollowed by 5 column volumes of Column Buffer C (50 mM MOPS pH 7.4, 10uM ZnSO₄, 0.1 mM CaCl₂, 1 mM dithiothreitol, and 2 mM benzamidine HCl).The enzyme was eluted into three successive pools. The first poolconsisted of enzyme from a 5 bed volume wash with Column Buffer Ccontaining 1 mM cAMP. The second pool consisted of enzyme from a 10 bedvolume wash with Column Buffer C containing 1 M NaCl. The final pool ofenzyme consisted of a 5 bed volume wash with Column Buffer C containing1 M NaCl and 20 mM cAMP.

[0161] The active pools of enzyme were collected and the cyclicnucleotide removed via conventional gel filtration chromatography orchromatography on hydroxy-apatite resins. Following removal of cyclicnucleotides, the enzyme pools were dialyzed against Dialysis Buffercontaining 25 mM MOPS pH 7.4, 10 μM ZnSO₄, 500 mM NaCl, 1 mM CaCl₂, 1 mMdithiothreitol, 1 mM benzamidine HCl, followed by dialysis againstDialysis buffer containing 50% glycerol. The enzyme was quick frozenwith the aid of dry ice and stored at −70° C.

[0162] The resultant preparations were about >90% pure by SDS-PAGE.These preparations had specific activities of about 0.1 to 1.0 μmol cAMPhydrolyzed per minute per milligram protein.

[0163] IC₅₀ Value Determinations

[0164] The parameter of interest in evaluating the potency of acompetitive enzyme inhibitor of PDE5 and/or PDE1c and PDE6 is theinhibition constant, i.e., K_(i). This parameter can be approximated bydetermining the IC₅₀, which is the inhibitor concentration that resultsin 50% enzyme inhibition, in a single dose-response experiment under thefollowing conditions.

[0165] The concentration of inhibitor is always much greater than theconcentration of enzyme, so that free inhibitor concentration (which isunknown) is approximated by total inhibitor concentration (which isknown).

[0166] A suitable range of inhibitor concentrations is chosen (i.e.,inhibitor concentrations at least several fold greater and several foldless than the K_(i) are present in the experiment). Typically, inhibitorconcentrations ranged from 10 nM to 10 μM.

[0167] The concentrations of enzyme and substrate are chosen such thatless than 20% of the substrate is consumed in the absence of inhibitor(providing, e.g., maximum substrate hydrolysis of from 10 to 15%), sothat enzyme activity is approximately constant throughout the assay.

[0168] The concentration of substrate is less than one-tenth theMichaelis constant (K_(m)). Under these conditions, the IC₅₀ willclosely approximate the K_(i). This is because of the Cheng-Prusoffequation relating these two parameters: IC₅₀=K_(i)(1+S/K_(m)), with(1+S/K_(m)) approximately 1 at low values of S/K_(m).

[0169] The IC₅₀ value is estimated from the data points by fitting thedata to a suitable model of the enzyme inhibitor interaction. When thisinteraction is known to involve simple competition of the inhibitor withthe substrate, a two-parameter model can be used:

Y=A/(1+x/B)

[0170] where the y is the enzyme activity measured at an inhibitorconcentration of x, A is the activity in the absence of inhibitor and Bis the IC₅₀. See Y. Cheng et al., Biochem. Pharmacol., 22:3099-3108(1973).

[0171] Effects of inhibitors of the present invention on enzymaticactivity of PDE5 and PDE6 preparations as described above were assessedin either of two assays which differed from each other principally onthe basis of scale and provided essentially the same results in terms ofIC₅₀ values. Both assays involved modification of the procedure of Wellset al., Biochim. Biophys. Acta, 384:430 (1975). The first of the assayswas performed in a total volume of 200 μl containing 50 mM Tris pH 7.5,3 mM Mg acetate, 1 mM EDTA, 50 μg/mL snake venom nucleotidase and 50 nM[³H]-cGMP (Amersham). Compounds of the invention were dissolved in DMSOfinally present at 2% in the assay. The assays were incubated for 30minutes at 30° C. and stopped by addition of 800 μl of 10 mM Tris pH7.5, 10 mM EDTA, 10 mM theophylline, 0.1 mM adenosine, and 0.1 mMguanosine. The mixtures were loaded on to 0.5 mL QAE Sephadex columns,and eluted with 2 mL of 0.1 M formate (pH 7.4). The eluted radioactivitywas measured by scintillation counting in Optiphase Hisafe 3.

[0172] A second, microplate, PDE assay was developed using Multiscreenplates and a vacuum manifold. The assay (100 μl) contained 50 mM Tris pH7.5, 5 mM Mg acetate, 1 mM EDTA and 250 μg/mL snake venom nucleotidase.The other components of the reaction mixture were as described above. Atthe end of the incubation, the total volume of the assays were loaded ona QAE Sephadex microcolumn plate by filtration. Free radioactivity waseluted with 200 μl of water from which 50 μl aliquots were analyzed byscintillation counting as described above.

[0173] The following examples are presented to further illustrate thepreparation of the claimed invention. The scope of the present inventionis not to be construed as merely consisting of the following examples.

EXAMPLE 1

[0174] The compound of structural formula (I) was prepared as describedin U.S. Pat. No. 5,859,006 and formulated in tablets using wetgranulation. Povidone was dissolved in water to make a 10% solution. Theactive compound, microcrystalline cellulose, croscarmellose sodium, andsodium lauryl sulfate were added to a high shear mixer and mixed for 2minutes. The powders were wet granulated with the povidone solution andextra water as required to complete the granulation. The resultantmixture was dried in a fluid bed drier with inlet air at 70° C±5° C.until the loss on drying was below 2.5%. The granules were passedthrough a Comil with a suitable screen (or a sieve) and added to asuitable mixer. The extragranular croscarmellose sodium and sodiumlauryl sulfate, and the colloidal anhydrous silica were passed through asuitable sieve (e.g., 500 micron) and added to the mixer and blended 5minutes. Magnesium stearate was added and blended for 2 minutes. Theblend was compressed to a target compression/weight of 250 mg using 9 mmround normal concave tooling.

[0175] The core tablets were coated with an aqueous suspension of OpadryOY-S-7322 using an Accelacota (or similar coating pan) using inlet airat 50° C. to 70° C. until the tablet weight was increased byapproximately 8 mg. Opadry OY-S-7322 containsmethylhydroxypropylcellulose Ph.Eur., titanium dioxide Ph. Eur.,Triacetin USP. Opadry increases the weight of each tablet to about 258mg. The amount of film coat applied per tablet may be less than thatstated depending on the process efficiency.

[0176] The tablets are filled into blister packs and accompanied bypackage insert describing the safety and efficacy of the compound.Formulations Component (mg per tablet) Selective PDE5 Inhibitor¹⁾ 1 5Hydroxypropylmethylcellulose 1 5 phthalate Microcrystalline Cellulose221.87 213.87 Croscarmellose Sodium 5.00 5.00 Sodium Lauryl Sulfate 2.502.50 Sulfate Povidone K30 9.38 9.38 Purified Water, USP (water q.s. q.s.for irrigation) Croscarmellose Sodium 5.00 5.00 Sodium Lauryl Sulfate2.50 2.50 Colloidal Anhydrous Silica 0.50 0.50 Magnesium Stearate 1.251.25 Total core subtotal 250.00 250.00 (Film coat Opadry OY-S-7322)about 8 mg about 8 mg

EXAMPLE 2

[0177] The following formula is used in preparing a finished dosage formcontaining 10 mg of the compound of structural formula (I). IngredientQuantity (mg) Granulation Selective PDE5 Inhibitor¹⁾ 10.00 LactoseMonohydrate 153.80 Lactose Monohydrate (spray dried) 25.00Hydroxypropylcellulose 4.00 Croscarmellose Sodium 9.00Hydroxypropylcellulose (EF) 1.75 Sodium Lauryl Sulfate 0.70 35.00Outside Powders Microcrystalline Cellulose (granular-102) 37.50Croscarmellose Sodium 7.00 Magnesium Stearate (vegetable) 1.25 Total 250mg

[0178] Purified Water, USP is used in the manufacture of the tablets.The water is removed during processing and minimal levels remain in thefinished product.

[0179] Tablets are manufactured using a wet granulation process. Astep-by-step description of the process is as follows. The drug andexcipients to be granulated are security sieved. The selective PDE5inhibitor is dry blended with lactose monohydrate (spray dried),hydroxypropylcellulose, croscarmellulose sodium, and lactosemonohydrate. The resulting powder blend is granulated with an aqueoussolution of hydroxypropylcellulose and sodium lauryl sulfate using aPowrex or other suitable high shear granulator. Additional water can beadded to reach the desired endpoint. A mill can be used to delump thewet granulation and facilitate drying. The wet granulation is driedusing either a fluid bed dryer or a drying oven. Once the material isdried, it can be sized to eliminate any large agglomerates.Microcrystalline cellulose, croscarmellose sodium, and magnesiumstearate are security sieved and added to the dry sized granules. Theseexcipients and the dry granulation are mixed until uniform using atumble bin, ribbon mixer, or other suitable mixing equipment. The mixingprocess can be separated into two phases. The microcrystallinecellulose, croscarmellose sodium, and the dried granulation are added tothe mixer and blended during the first phase, followed by the additionof the magnesium stearate to this granulation and a second mixing phase.

[0180] The mixed granulation then is compressed into tablets using arotary compression machine. The core tablets are film coated with anaqueous suspension of the appropriate color mixture in a coating pan(e.g., Accela Cota). The coated tablets can be lightly dusted with talcto improve tablet handling characteristics.

[0181] The tablets are filled into plastic containers (30tablets/container) and accompanied by package insert describing thesafety and efficacy of the compound.

EXAMPLE 3

[0182] The following formula is used in preparing a finished dosage formof 5 mg of the compound of structural formula (I). Ingredient Quantity(mg) Granulation Selective PDE5 Inhibitor¹⁾ 2.50 Lactose Monohydrate79.395 Lactose Monohydrate (spray dried) 12.50 Hydroxypropylcellulose2.00 Croscarmellose Sodium 4.50 Hydroxypropylcellulose (EF) 0.875 SodiumLauryl Sulfate 0.35 Outside Powders Microcrystalline Cellulose(granular-102) 18.75 Croscarmellose Sodium 3.50 Magnesium Stearate(vegetable) 0.63 Total 125 mg

[0183] The dosage form of Example 3 was prepared in an identical mannerto the dosage form of Example 2.

EXAMPLE 4

[0184] Solution Capsule Ingredient mg/capsule Percent (%) Selective PDE5Inhibitor¹⁾ 10 2 PEG400 NF 490 98 Fill Weight 500 100

[0185] The gelatin capsules are precisely filled by pumping an accuratefill volume of pre-dissolved drug formulation into the partially sealedcavity of a capsule. Immediately following injection fill of the drugsolution formulation, the capsule is completely heat sealed.

[0186] The capsules are filled into plastic containers and accompaniedby a package insert.

EXAMPLE 5

[0187] This study was a randomized, double-blind, placebo-controlled,two-way crossover design clinical pharmacology drug interaction studythat evaluated the hemodynamic effects of concomitant administration ofa selective PDE5 inhibitor Study Drug (i.e., the compound of structuralformula (I)) and short-acting nitrates on healthy male volunteers. Inthis study, the subjects received either the Study Drug at a dose of 10mg or a placebo, daily for seven days. On the sixth or seventh day, thesubjects received sublingual nitroglycerin (0.4 mg) while supine on atilt table. The nitroglycerin was administered 3 hours after Study Drugdosing, and all subjects kept the nitroglycerin tablet under theirtongue until it completely dissolved. The subjects were tilted to 700head-up every 5 minutes for a total of 30 minutes with measurement ofblood pressure and heart rate. There were no discontinuations among thetwenty-two healthy male subjects (ages 19 to 60 years old) that enteredthis study.

[0188] In a preliminary analysis of this study, the Study Drug was welltolerated and there were no serious adverse events. There were no StudyDrug-associated changes in laboratory safety assessments or 12-leadECGs. The most common adverse events were headache, dyspepsia, and backpain. The study demonstrated minimal effects on mean systolic bloodpressure and on mean maximal nitroglycerin-induced decrease in systolicblood pressure and the maximal nitroglycerin-induced decrease insystolic blood pressure among all patients.

EXAMPLE 6

[0189] In two randomized, double-blinded placebo controlled studies, thecompound of structural formula (I), at a range of doses in both dailydosing and for on demand therapy for sexual encounters and intercoursein the home setting, was administered to patients in need thereof. Dosesfrom 5 to 20 mg of the compound of structural formula (I) wereefficacious and demonstrated no flushing and no reports of visionabnormalities. It was found that a 10 mg dose of the compound ofstructural formula (I) was fully efficacious and demonstrated minimalside effects (no flushing and no reports of blue vision).

[0190] Erectile function was assessed by the International Index ofErectile Function (IIEF) (Rosen et al., Urology, 49, pp. 822-830(1997)), diaries of sexual attempts, and a global satisfaction question.The compound of structural formula (I) significantly improved erectilefunction as assessed by all endpoints. In both “on demand” and dailydose regimens, the compound of structural formula (I) significantlyimproved erectile function in doses between 1 and 20 mg.

EXAMPLE 7

[0191] A third clinical study was a randomized, double-blind,placebo-controlled study using a compound of structural formula (I)(Study Drug) administered “on demand” to patients with male erectiledysfunction. The Study Drug was administered over a period of eightweeks in the treatment of male erectile dysfunction (ED). Erectiledysfunction (ED) is defined as the persistent inability to attain and/ormaintain an erection adequate to permit satisfactory sexual performance.“On demand” dosing is defined as intermittent administration of StudyDrug prior to expected sexual activity.

[0192] The study population consisted of 212 men, at least 18 years ofage, with mild to severe erectile dysfunction. The Study Drug was orallyadministered as tablets of coprecipitate made in accordance with ButlerU.S. Pat. No. 5,985,326. The Study Drug was administered in 2 mg, 5 mg,10 mg, and 25 mg doses, “on demand” and not more than once every 24hours. Treatment with all nitrates, azole antifungals (e.g.,ketoconazole or itraconazole), warfarin, erythromycin, or antiandrogenswas not allowed at any time during the study. No other approved orexperimental medications, treatments, or devices used to treat ED wereallowed. Forty-one subjects were administered a placebo.

[0193] The two primary efficacy variables were the ability of a subjectto penetrate his partner and his ability to maintain an erection duringintercourse, as measured by the International Index of Erectile Function(IIEF). The IIEF Questionnaire contains fifteen questions, and is abrief, reliable measure of erectile function. See R. C. Rosen et al.,Urology, 49, pp. 822-830 (1997).

[0194] Secondary efficacy variables were IIEF domain scores for erectilefunction, orgasmic function, sexual desire, intercourse satisfaction,and overall satisfaction; the patient's ability to achieve an erection,ability to insert his penis into his partner's vagina, completion ofintercourse with ejaculation, satisfaction with the hardness of hiserection, and overall satisfaction, all as measured by the SexualEncounter Profile (SEP) diary; and a global assessment question asked atthe end of the treatment period. The SEP is a patient diary instrumentdocumenting each sexual encounter during the course of the study.

[0195] The safety analysis of the study included all enrolled subjects,and was assessed by evaluating all reported adverse events, and changesin clinical laboratory values, vital signs, physical examinationresults, and electrocardiogram results.

[0196] At endpoint, patients who rated their penetration ability (IIEFQuestion 3) as “almost always or always” were as follows: 17.5% in theplacebo group, 38.1% in the 2 mg group, 48.8% in the mg group, 51.2% inthe 10 mg group, and 83.7% in the 25 mg group. Comparisons revealedstatistically significant differences in change in penetration abilitybetween placebo and all dose levels of the Study Drug.

[0197] At endpoint, patients who rated their ability to maintain anerection (IIEF Question 4) during intercourse as “almost always oralways” are as follows: 10.0% in the placebo group, 19.5% in the 2 mggroup, 32.6% in the 5 mg group, 39.0% in the 10 mg group, and 69.0% inthe 25 mg group. Comparison revealed statistically significantdifferences in change in penetration ability between placebo and thethree higher dose levels of Study Drug.

[0198] Overall, this study demonstrated that all four doses of StudyDrug, namely 2 mg, 5 mg, 10 mg, and 25 mg, taken “on demand” producedsignificant improvement, relative to placebo, in the sexual performanceof men with erectile dysfunction as assessed by the IIEF, by patientdiaries assessing frequency of successful intercourse and intercoursesatisfaction, and by a global assessment. This improvement wasdemonstrated in a broad study population that included patients whoexhibited all severities of erectile dysfunction. Most adverse eventswere mild or moderate in severity. Significantly, no adverse eventsrelated to color vision disturbances were reported by any patient.

[0199] The combined results from clinical studies showed thatadministration of a compound of structural formula (I) effectivelytreats male erectile dysfunction, as illustrated in the following table.IIEF ERECTILE FUNCTION DOMAIN (Change from Baseline) Unit Dose n Mean ±SD p placebo 131 0.8 ± 5.3  2 mg 75 3.9 ± 6.1 <.001  5 mg 79 6.6 ± 7.1<.001 10 mg 135 7.9 ± 6.7 <.001 25 mg 132 9.4 ± 7.0 <.001 50 mg 52 9.8 ±5.5 <.001 100 mg  49 8.4 ± 6.1 <.001

[0200] However, it also was observed from the combined clinical studiesthat the percent of treatment-emergent adverse events increased with anincreasing unit dose of the compound of structural formula (I), asillustrated in the following table. Treatment-Emergent Adverse Events(%) Unit Dose (mg) Event Placebo 2 5 10 25 50 100 Headache 10 12 10 2329 34 46 Dyspepsia 6 3 14 13 19 20 25 Back Pain 5 3 3 15 18 24 22Myalgia 3 0 3 9 16 20 29 Rhinitis 3 7 3 4 4 0 2 Conjunctivitis 1 0 1 1 02 5 Eyelid Edema 0 0 0 1 1 2 3 Flushing 0 0 0 <1 0 3 7 Vision 0 0 0 0 00 0 Abnormalities

[0201] The above table shows an increase in adverse events at 25 mgthrough 100 mg unit doses. Accordingly, even though efficacy in thetreatment of ED was observed at 25 mg to 100 mg doses, the adverseevents observed from 25 mg to 100 mg doses must be considered.

[0202] In accordance with the present invention, a unit dose of about 1to about 20 mg, preferably about 2 to about 20 mg, more preferably about5 to about 20 mg, and most preferably about 5 to about 15 mg,administered up to a maximum of 20 mg per 24-hour period, botheffectively treats ED and minimizes or eliminates the occurrence ofadverse side effects. Importantly, no vision abnormalities were reportedand flushing was essentially eliminated. Surprisingly, in addition totreating ED in individuals, with about 1 to about 20 mg unit dose of thecompound of structural formula (I), with a minimum of adverse sideeffects, individuals undergoing nitrate therapy also can be treated forED by the method and composition of the present invention.

[0203] The principles, preferred embodiments, and modes of operation ofthe present invention have been described in the foregoingspecification. The invention intended to be protected herein, however,is not construed to be limited to the particular forms disclosed,because they are to be regarded as illustrative rather than restrictive.Variations and changes may be made by those skilled in the art withoutdeparting from the spirit of the invention.

What is claimed is:
 1. An article of manufacture for humanpharmaceutical use comprising: (a) an oral dosage form comprising about1 to about 20 mg of a selective PDE5 inhibitor having (i) at least a 100fold differential in IC₅₀ values for the inhibition of PDE5 versus PDE6,(ii) at least a 1000 fold differential in IC₅₀ values for the inhibitionof PDE5 versus PDE1c, (iii) an IC₅₀ for the inhibition of PDE5 less than10 nM, and (iv) sufficient bioavailability to be effective in about 1 toabout 20 mg unit oral dosages; (b) a package insert providing that thePDE5 inhibitor is useful to treat sexual dysfunction in a patient inneed thereof, and that is free of contradictions associated withadministration of organic nitrates; and (c) a container.
 2. An articleof manufacture for human pharmaceutical use comprising: (a) an oraldosage form comprising about 1 to about 20 mg of selective PDE5inhibitor having (i) at least a 100 fold differential in IC₅₀ values forthe inhibition of PDE5 versus PDE6, (ii) at least a 1000 folddifferential in IC₅₀ values for the inhibition of PDE5 versus PDE1c,(iii) an IC₅₀ less than 10 nM, and (iv) a sufficient bioavailability tobe effective in about 1 to about 20 mg unit oral dosages; (b) a packageinsert providing that the PDE5 inhibitor is useful to treat sexualdysfunction in a patient in need thereof and that is using an organicnitrate; and (c) a container.
 3. An article of manufacture for humanpharmaceutical use comprising: (a) an oral dosage form comprising about1 to about 20 mg of a selective PDE5 inhibitor having (i) at least a 100fold differential in IC₅₀ values for the inhibition of PDE5 versus PDE6,(ii) at least 1000 fold differential in IC₅₀ values for the inhibitionof PDE5 versus PDE1c, (iii) an IC₅₀ less than 10 nM, and (iv) asufficient bioavailability to be effective in about 1 to about 20 mgunit oral dosages; (b) a package insert providing that the PDE5inhibitor is useful to treat sexual dysfunction in a patient in needthereof and that is suffering from a condition selected from the groupconsisting of a retinal disease, proneness to flushing, proneness tovision abnormalities, class 1 congestive heart failure, a myocardialinfarction 90 days or more before onset of the sexual dysfunctiontreatment, and combinations thereof; and (c) a container.
 4. The articleof claim 3 wherein the retinal disease is diabetic retinopathy orretinitis pigmentosa.
 5. The article of claim 3 wherein said packageinsert reports that incidences of flushing are less than 2% of treatedpatients.
 6. The article of claims 1 through 5 wherein the oral dosageform comprises about 5 mg, about 10 mg, or about 20 mg, of a selectivePDE5 inhibitor.
 7. The article of claims 1 through 5 wherein the packageinsert provides a maximum dosage of the selective PDE5 inhibitor ofabout 20 mg per 24-hour period.
 8. The article of claims 1 through 5,wherein the selective PDE5 inhibitor has the structure


9. A method of treating sexual dysfunction comprising using an articleof manufacture of claims 1 through
 5. 10. A method of treating sexualdysfunction in a patient being treated with an organic nitratecomprising administration of an oral dosage form of a PDE5 inhibitor inan amount of about 1 to about 20 mg.
 11. A method of treating sexualdysfunction in a patient prone to vision abnormalities, prone toflushing, suffering from a retinal disease, suffering from class 1congestive heart failure, or that suffered a myocardial infarction 90days or more prior to onset of the sexual dysfunction treatmentcomprising administration of an oral dosage form of a PDE5 inhibitor inan amount of about 1 to about 20 mg.